The broad objective of this project is to understand the relationship between normal tissue stem cells and cancer stem cells in mouse models of mammary cancer. The cancer stem cell hypothesis proposes that a small population of tumor-initiating cancer stem cells (T-IC) exists within the heterogeneous population of cells in a malignancy. Normal stem cells or multipotent progenitor cells of the tissue in which the primary cancer arises are likely precursors of T-IC. Limitations on the definition and isolation of stem cells are a major obstacle to understanding T-IC. Parity-induced mammary epithelial cells (PI-MEC) are a self- renewing, pluripotent population, thus fulfilling the functions of mammary stem or progenitor cells. PI-MEC involvement in mammary cancer was reported, but a method to isolate pure populations of PI-MEC was not described. Fluorescently marked PI-MEC cells could be purified and experimentally manipulated. Mammary tumors and preneoplastic changes are parity-induced in the C57BL/6J-Tg(WapTAg)1Knw (Waptag 1) mouse. Thus Waptag 1 is an attractive model in which to test the potential of fluorescently marked PI-MECs as mammary tumor stem cells. To test the hypothesis that mammary tumors in Waptag 1 arise from PI-MEC cells, this project will develop two novel mouse models. The first will be derived from intercrossing B6-Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J mice with B6-Cg-Tg(WapCre)11738Mam/J mice. PI- MEC will express enhanced green fluorescent protein in this unique model. The second model will be derived from crossing the first with Waptag 1 mice. The properties of PI-MEC will be characterized in vivo using transplantation studies of mammary epithelial cells isolated by fluorescence activated cell sorting from control glands and from glands of mice at different stages in Waptag 1 tumorigenesis. Relevance to Public Health. Development of a mouse model of mammary cancer in which a defined cellular subpopulation (PI-MEC) with properties of stem cells can be isolated and analyzed will enable (i) definition of the immunophenotype of different mammary tumor stem cells, the gold standard of precise identification;(ii) investigation of molecular events in the very earliest stages of mammary cancer. Extending the use of fluorescently marked PI-MEC to multiple mouse mammary tumor models will be important to understand the general extent to which this cell population contributes to different types of mammary cancer.